The proposed studies will optimize mechanical stimuli applied to mesenchymal stem cell (MSC) constructs to test the global hypothesis that delivering in vivo strain simulations boths stiffens repairs and shortens healing time. This research addresses 3 important clinical problems: filling tendon defects with biological constructs to reduce pain and stiffness and to provide grafts for revision surgery;repairing large rotator cuff tears and augmenting ligament reconstructions;and repairing chronic degenerative tendon injuries. Using a functional tissue engineering or FTE roadmap with linked in vitro and in vivo studies, we will establish in vivo forces and displacements for 4 activities of daily living in the rabbit patellar tendon model (PT) (Aim 1). Using a novel design strategy, 6 mechanical treatment factors will then be varied in culture to optimize the construct's tangent stiffness before surgery (Aim 2). Constructs with significantly different tangent stiffness values will be implanted in rabbit PT defects to examine significant improvements in repair stiffness (Aim 3). We will thus test a series of related hypotheses: Hyp 1. The rabbit PT maintains non-zero tissue strain and force for all activities. Hyp 2. Increasing the grade or inclination of the gait surface increases in vivo force and strain parameters. Hyp 3. Rabbit and mouse constructs exposed to in vivo strain shapes develop higher tangent stiffness than constructs exposed to trapezoidal or sinusoidal profiles. Hyp 4. Increasing strain amplitude, within physiological limits, increases tangent stiffness in culture. Hyp. 5. Strain patterns with similar times to rise and fall and similar periods as in vivo signals exhibit the largest stiffness values in culture. Hyp 6. Mouse constructs having the largest tangent stiffness in culture exhibit the greatest increases in col I gene expression. Hyp 7. Increasing construct stiffness in culture increases repair stiffness after surgery and better aligns cells and extracellular matrix in the repair site. Our novel, interactive in vitro / in vivo study should help to develop in vitro predictors of in vivo repair outcome after surgery that also apply to other tissue systems.